Guthrie / Weissman Guide to Yeast Genetics: Functional Genomics, Proteomics, and Other Systems Analysis

2. Auflage 2010
ISBN: 978-0-12-375173-7
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, 961 Seiten

This fully updated edition of the bestselling three-part Methods in Enzymology series, Guide to Yeast Genetics and Molecular Cell Biology is specifically designed to meet the needs of graduate students, postdoctoral students, and researchers by providing all the up-to-date methods necessary to study genes in yeast. Procedures are included that enable newcomers to set up a yeast laboratory and to master basic manipulations. This volume serves as an essential reference for any beginning or experienced researcher in the field.
Provides up-to-date methods necessary to study genes in yeast.
Includes proceedures that enable newcomers to set up a yeast laboratory and to master basic manipulations.
This volume serves as an essential reference for any beginning or experienced researcher in the field.

Autoren/Hrsg.

Guthrie, Christine

Weissman, Jonathan

Inhaltsverzeichnis

1;Front Cover;1
2;Methods in Enzymology;4
3;Copyright Page;5
4;Contents;6
5;Contributors;16
6;Preface;24
7;Volumes in Series;26
8;Section 1: Functional Genomics;54
8.1;Chapter 1: Analysis of Gene Function Using DNA Microarrays;56
8.1.1;1. Introduction and Experimental Design;57
8.1.1.1;1.1. Single-mutant analysis;57
8.1.1.2;1.2. Double-mutant analysis;59
8.1.2;2. Methods;61
8.1.2.1;2.1. Experimental design;61
8.1.2.2;2.2. Cell growth;62
8.1.2.3;2.3. Total RNA isolation and purification;62
8.1.2.4;2.4. Purification of poly-A RNA;63
8.1.2.5;2.5. Reverse transcription and dye labeling;64
8.1.2.6;2.6. Hybridization;65
8.1.2.7;2.7. Microarray washing;66
8.1.2.8;2.8. Array scanning;67
8.1.2.9;2.9. Gridding and normalization;67
8.1.3;References;69
8.2;Chapter 2: An Introduction to Microarray Data Analysis and Visualization;72
8.2.1;1. Introduction;73
8.2.1.1;1.1. Overview;73
8.2.1.2;1.2. Commonly used terms in microarray data analysis;75
8.2.1.3;1.3. A simple case study;76
8.2.2;2. Experimental Design: Single-Sample Versus Competitive Hybridization;76
8.2.2.1;2.1. Single-sample hybridization;77
8.2.2.2;2.2. Competitive hybridization;77
8.2.2.3;2.3. Choosing the best approach;78
8.2.3;3. Image Analysis;79
8.2.3.1;3.1. What is a digital image?;80
8.2.3.2;3.2. Data files;80
8.2.3.3;3.3. Software tools;81
8.2.4;4. Preprocessing;82
8.2.4.1;4.1. Software tools;82
8.2.4.2;4.2. Calculating ratio values;85
8.2.4.3;4.3. Normalizing ratio values;86
8.2.4.4;4.4. Quality assessment, filtering, and handling replicates;89
8.2.4.5;4.5. Preprocessing Affymetrix arrays;90
8.2.5;5. Visualizing Data Using Cluster Analysis;91
8.2.5.1;5.1. Hierarchical clustering;91
8.2.5.2;5.2. Partitioning and network-based approaches;93
8.2.6;6. Assessing the Statistical Evidence for Differential Expression;94
8.2.6.1;6.1. Significance analysis of microarrays;94
8.2.6.2;6.2. Limma;95
8.2.7;7. Exploring Gene Sets;95
8.2.7.1;7.1. Gene Ontology term mapping;95
8.2.7.2;7.2. Motif searching;96
8.2.7.3;7.3. Network visualization;96
8.2.7.4;7.4. Graphing array data on genome tracks;96
8.2.8;8. Managing Data;97
8.2.8.1;8.1. Data persistence and integrity;97
8.2.8.2;8.2. Public data repositories and MIAME compliance;99
8.2.9;Acknowledgments;102
8.2.10;References;102
8.3;Chapter 3: Genome-Wide Approaches to Monitor Pre-mRNA Splicing;104
8.3.1;1. Introduction;105
8.3.2;2. Microarray Design;106
8.3.3;3. Sample Preparation;109
8.3.3.1;3.1. Cell collection;109
8.3.3.2;3.2. RNA isolation;110
8.3.3.3;3.3. cDNA synthesis;113
8.3.3.4;3.4. Fluorescent labeling of cDNA;117
8.3.3.5;3.5. Microarray hybridization;119
8.3.3.6;3.6. Microarray washing;120
8.3.4;4. Microarray Data Collection;121
8.3.5;5. Microarray Data Analysis;122
8.3.5.1;5.1. Data normalization;123
8.3.5.2;5.2. Replication;123
8.3.5.3;5.3. Splicing specific data;124
8.3.5.4;5.4. Extracting biological meaning;124
8.3.6;6. Future Methodologies;125
8.3.7;Acknowledgments;126
8.3.8;References;127
8.4;Chapter 4: ChIP-Seq: Using High-Throughput DNA Sequencing for Genome-Wide Identification of Transcription Factor Binding Site;130
8.4.1;1. Introduction;131
8.4.2;2. Protocols;134
8.4.2.1;2.1. Chromatin immunoprecipitation;134
8.4.2.2;2.2. Input DNA preparation;138
8.4.2.3;2.3. Illumina sequencing DNA library generation;139
8.4.2.4;2.4. Barcode design and adapter annealing;144
8.4.2.5;2.5. Illumina sequencing;146
8.4.3;3. Sequencing Data Management;146
8.4.4;4. Genome Analysis Pipeline;147
8.4.5;5. Examining Data Quality and Parsing Barcoded Data;148
8.4.6;6. Visualization in Genome Browser;148
8.4.6.1;6.1. Low-level analysis;149
8.4.6.2;6.2. High-level analysis;154
8.4.6.3;6.3. Troubleshooting;154
8.4.7;7. Conclusion and Future Directions;155
8.4.8;Acknowledgments;155
8.4.9;References;155
8.5;Chapter 5: Genome-Wide Mapping of Nucleosomes in Yeast;158
8.5.1;1. Introduction;158
8.5.2;2. Isolation of Mononucleosomal DNA;160
8.5.3;3. Variation in Titration Level Used for Nucleosome Purification;165
8.5.4;4. Labeling of Mononucleosomal DNA for Tiling Microarray Analysis;166
8.5.4.1;4.1. Protocol;167
8.5.5;5. Generation of Nucleosomal DNA Libraries for Deep Sequencing;168
8.5.6;References;170
8.6;Chapter 6: Genome-Wide Translational Profiling by Ribosome Footprinting;172
8.6.1;1. Introduction;173
8.6.2;2. Ribosome Footprint Generation and Purification;174
8.6.2.1;2.1. Extract preparation;175
8.6.2.2;2.2. Nuclease digestion and monosome purification;176
8.6.2.3;2.3. Footprint fragment purification;177
8.6.2.4;2.4. Fragmented mRNA preparation;179
8.6.3;3. Sequencing Library Preparation;181
8.6.3.1;3.1. Polyadenylation;182
8.6.3.2;3.2. Reverse transcription;183
8.6.3.3;3.3. Circularization;183
8.6.3.4;3.4. PCR amplification;184
8.6.4;4. Data Analysis;186
8.6.4.1;4.1. Mapping polyadenylated sequences;187
8.6.4.2;4.2. Reference databases;188
8.6.4.3;4.3. Selecting high-quality alignments;189
8.6.4.4;4.4. Quantifying gene expression;189
8.6.5;5. Solutions and Common Procedures;191
8.6.5.1;5.1. Solutions;191
8.6.5.2;5.2. Oligonucleotides;192
8.6.5.3;5.3. Nucleic acid precipitation;192
8.6.5.4;5.4. Nucleic acid gel extraction;193
8.6.6;Acknowledgments;193
8.6.7;References;193
9;Section 2: Systematic Genetic Analysis;196
9.1;Chapter 7: Synthetic Genetic Array (SGA) Analysis in Saccharomyces cerevisiae and Schizosaccharomyces pombe;198
9.1.1;1. Introduction;199
9.1.2;2. Methodology;201
9.1.2.1;2.1. SGA query strain construction;201
9.1.2.2;2.2. Pin tool sterilization procedures;203
9.1.2.3;2.3. Constructing a 1536-density DMA;205
9.1.2.4;2.4. SGA procedure;206
9.1.2.5;2.5. Double mutant array image acquisition and processing;208
9.1.2.6;2.6. Quantitative scoring of genetic interactions using colony size-based fitness measurements;210
9.1.2.7;2.7. Interpretation and analysis of genetic interactions;212
9.1.2.8;2.8. S. pombe SGA;218
9.1.3;3. Media and Stock Solutions;221
9.1.3.1;3.1. SGA media and stock solutions;221
9.1.3.2;3.2. SpSGA media and stock solutions;224
9.1.4;4. Applications of SGA Methodology;224
9.1.4.1;4.1. Integrating SGA and high-content screening;224
9.1.4.2;4.2. Essential gene and higher order genetic interactions;227
9.1.4.3;4.3. Combining SGA and gene overexpression libraries;227
9.1.4.4;4.4. Applying SGA as a method for high-resolution genetic mapping (SGAM);228
9.1.4.5;4.5. Chemical genomics;228
9.1.5;References;229
9.2;Chapter 8: Making Temperature-Sensitive Mutants;234
9.2.1;1. Introduction;235
9.2.2;2. Diploid Shuffle-Plasmid Method;236
9.2.2.1;2.1. General description of the diploid shuffle—plasmid method;236
9.2.2.2;2.2. Materials;238
9.2.2.3;2.3. Methods;239
9.2.3;3. Diploid Shuffle-Chromosome Method;247
9.2.3.1;3.1. A general description;247
9.2.3.2;3.2. Materials;249
9.2.3.3;3.3. Methods;251
9.2.4;4. Perspectives;255
9.2.5;References;256
9.3;Chapter 9: Quantitative Genetic Interaction Mapping Using the E-MAP Approach;258
9.3.1;1. Introduction;259
9.3.2;2. Selection of Mutations for Genetic Analysis;262
9.3.3;3. Generation and Measurement of Double Mutant Strains;264
9.3.3.1;3.1. Basic SGA protocol;267
9.3.3.2;3.2. Basic PEM protocol;272
9.3.3.3;3.3. Digital photography;274
9.3.4;4. Data Processing and Computation of Scores;274
9.3.4.1;4.1. Preprocessing and normalization;274
9.3.4.2;4.2. Computing expected colony sizes;276
9.3.4.3;4.3. Computing genetic interaction scores;276
9.3.4.4;4.4. Quality control;277
9.3.5;5. Extraction of Biological Hypotheses;277
9.3.5.1;5.1. Identifying genes acting in the same pathway using patterns of interactions;277
9.3.5.2;5.2. Using individual interactions to predict enzyme–substrate relationships;278
9.3.5.3;5.3. Using individual interactions to predict opposing enzyme relationships;280
9.3.5.4;5.4. Dissecting multiple roles of a single gene by detailed comparison of interaction patterns;281
9.3.6;6. Perspective;282
9.3.7;References;282
9.4;Chapter 10: Exploring Gene Function and Drug Action Using Chemogenomic Dosage Assays;286
9.4.1;1. Introduction;287
9.4.2;2. Methodology;290
9.4.2.1;2.1. Yeast deletion strain pool and MSP pool construction;290
9.4.2.2;2.2. Determining the drug dosage;291
9.4.2.3;2.3. Experimental pool growth for deletion and overexpression collections;293
9.4.2.4;2.4. Purification and amplification of barcodes and ORFs;296
9.4.2.5;2.5. Hybridization;298
9.4.2.6;2.6. Analysis of results;300
9.4.2.7;2.7. Confirmation of microarray data;303
9.4.3;3. Experimental Considerations;305
9.4.4;4. Perspectives;306
9.4.5;Acknowledgments;307
9.4.6;References;307
10;Section 3: Proteomics;310
10.1;Chapter 11: Yeast Expression Proteomics by High-Resolution Mass Spectrometry;312
10.1.1;1. Introduction;313
10.1.2;2. Background, Methods, and Applications;314
10.1.2.1;2.1. The challenge;314
10.1.2.2;2.2. Background on MS instrumentation for ‘‘shotgun’’ proteomics;316
10.1.2.3;2.3. Quantitative proteomics;320
10.1.2.4;2.4. Computational proteomics and data ana;323
10.1.2.5;2.5. Perspective and outlook;323
10.1.3;3. Protocols;325
10.1.3.1;3.1. Yeast strains for SILAC proteomics experiments;325
10.1.3.2;3.2. Media for SILAC labeling;326
10.1.3.3;3.3. Growing yeast cultures for SILAC labeling;327
10.1.3.4;3.4. Extract Preparation for SILAC experiments;327
10.1.3.5;3.5. In-solution digest of proteins for MS;327
10.1.3.6;3.6. Test of label incorporation;327
10.1.3.7;3.7. Peptide IEF (Optional);328
10.1.3.8;3.8. MS analysis;329
10.1.3.9;3.9. Identification and quantitation of peptides and proteins;330
10.1.4;Acknowledgments;331
10.1.5;References;331
10.2;Chapter 12: High-Quality Binary Interactome Mapping;334
10.2.1;1. Introduction;335
10.2.2;2. High-Quality Binary Interactome Mapping;336
10.2.2.1;2.1. Production and verification of Y2H datasets;338
10.2.2.2;2.2. Validation of Y2H datasets to produce reliable binary interactome maps;341
10.2.2.3;2.3. Biological evaluation of binary interactome maps;344
10.2.3;3. High-Throughput Y2H Pipeline;345
10.2.3.1;3.1. Assembly of DB-X and AD-Y expression plasmids;345
10.2.3.2;3.2. Yeast transformation;349
10.2.3.3;3.3. Autoactivator removal and AD-Y pooling;351
10.2.3.4;3.4. Screening and phenotyping;354
10.2.3.5;3.5. Verification;358
10.2.3.6;3.6. Media and plates;360
10.2.4;4. Validation Using Orthogonal Binary Interaction Assays;362
10.2.5;5. Conclusion;365
10.2.6;Acknowledgments;365
10.2.7;References;366
10.3;Chapter 13: Quantitative Analysis of Protein Phosphorylation on a System-Wide Scale by Mass Spectrometry-Based Proteomics;370
10.3.1;1. Introduction;371
10.3.2;2. Protocols;372
10.3.2.1;2.1. Generation of peptide samples;372
10.3.2.2;2.2. Phosphopeptide isolation;375
10.3.2.3;2.3. Mass spectrometric analyses of the phosphopeptide isolates;379
10.3.2.4;2.4. Data analyses;382
10.3.3;Acknowledgments;384
10.3.4;References;385
10.4;Chapter 14: A Toolkit of Protein-Fragment Complementation Assays for Studying and Dissecting Large-Scale and Dynamic Protein-Protein Interactions in Living Cells;388
10.4.1;1. Introduction;389
10.4.2;2. General Considerations in Using PCA;390
10.4.3;3. DHFR PCA Survival-Selection for Large-Scale Analysis of PPIs;392
10.4.3.1;3.1. Materials;394
10.4.3.2;3.2. Procedure;394
10.4.4;4. A Life and Death Selection PCA Based on the Prodrug-Converting Cytosine Deaminase for Dissection of PPIs;401
10.4.4.1;4.1. Preparation for a two-step OyCD PCA screen;403
10.4.4.2;4.2. Materials;403
10.4.4.3;4.3. Procedure;405
10.4.5;5. Visualizing the Localization of PPIs with GFP Family Fluorescent Protein PCAs;409
10.4.5.1;5.1. Materials;411
10.4.5.2;5.2. Procedure;412
10.4.6;6. Studying Dynamics of PPIs with Luciferase Reporter PCAs;414
10.4.6.1;6.1. Materials;415
10.4.6.2;6.2. Procedure;416
10.4.7;References;419
10.5;Chapter 15: Yeast Lipid Analysis and Quantification by Mass Spectrometry;422
10.5.1;1. Introduction;423
10.5.2;2. Methods;426
10.5.2.1;2.1. Sample preparation;426
10.5.2.2;2.2. Normalization for starting amount of material;428
10.5.2.3;2.3. Lipid extraction (for glycerophospholipids and sphingolipids);428
10.5.2.4;2.4. Sterol isolation;429
10.5.2.5;2.5. Lipid analysis;430
10.5.3;Acknowledgments;442
10.5.4;References;442
10.6;Chapter 16: Mass Spectrometry-Based Metabolomics of Yeast;446
10.6.1;1. Introduction;447
10.6.2;2. LC-MS Basics;448
10.6.3;3. Experimental Design;448
10.6.4;4. Strains;451
10.6.5;5. Culture Conditions;451
10.6.5.1;5.1. Batch liquid culture and chemostats;451
10.6.5.2;5.2. Protocol for harvesting yeast by vacuum filtration;453
10.6.5.3;5.3. Protocol for harvesting yeast by centrifugation after methanol quenching;453
10.6.5.4;5.4. Filter culture;454
10.6.5.5;5.5. Protocol for growth of yeast on filters atop agarose support;455
10.6.6;6. Metabolite Extraction;455
10.6.6.1;6.1. Extraction protocol for cells on filters (from vacuum filtration of liquid culture, or from filter cultures);457
10.6.6.2;6.2. Extraction protocol for cells after methanol quenching;457
10.6.7;7. Chemical Derivatization of Metabolites;458
10.6.7.1;7.1. Amino acid derivatization;459
10.6.7.2;7.2. Thiol and disulfide derivatiz;459
10.6.8;8. LC-MS for Mixture Analysis;459
10.6.9;9. Liquid Chromatography;460
10.6.10;10. Electrospray Ionization;463
10.6.11;11. Mass Spectrometry;464
10.6.11.1;11.1. Triple quadrupole mass spectrometers;464
10.6.11.2;11.2. High-resolution mass analyzers;466
10.6.11.3;11.3. Hybrid instruments;468
10.6.12;12. Targeted Data Analysis;468
10.6.13;13. Untargeted Data Analysis;471
10.6.13.1;13.1. Adducts;471
10.6.13.2;13.2. Isotopic variants;473
10.6.13.3;13.3. In-source fragmentation;474
10.6.13.4;13.4. Unknown identification;474
10.6.14;14. Future Outlook;474
10.6.15;References;475
11;Section 4: Systems Analysis;480
11.1;Chapter 17: Imaging Single mRNA Molecules in Yeast;482
11.1.1;1. Introduction;482
11.1.2;2. RNA FISH Protocol;485
11.1.2.1;2.1. Designing oligonucleotides;485
11.1.2.2;2.2. Coupling fluorophores to oligonucleotides;486
11.1.2.3;2.3. Purification of probes using HPLC;488
11.1.2.4;2.4. Fixing S. cerevisiae;490
11.1.2.5;2.5. Hybridizing probes to target mRNA;491
11.1.2.6;2.6. Image processing: Detecting diffraction-limited mRNA spot;495
11.1.3;3. Example: STL1 mRNA Detection in Response to NaCl Shock;497
11.1.4;4. Conclusions;498
11.1.5;Acknowledgments;498
11.1.6;References;498
11.2;Chapter 18: Reconstructing Gene Histories in Ascomycota Fungi;500
11.2.1;1. Introduction;501
11.2.2;2. Synergy;504
11.2.2.1;2.1. Overview;504
11.2.2.2;2.2. Defining orthogroups;504
11.2.2.3;2.3. Scoring gene similarity;506
11.2.2.4;2.4. Gene similarity graph;507
11.2.2.5;2.5. Identifying orthogroups;507
11.2.3;3. Evaluating Orthogroup Quality;512
11.2.3.1;3.1. Fungal orthogroup robustness;514
11.2.3.2;3.2. Comparison to curated resource;516
11.2.3.3;3.3. Simulated orthogroups;518
11.2.4;4.Biological Analysis of Gene Histories;519
11.2.4.1;4.1. Defining orthogroup categories;519
11.2.4.2;4.2. Singletons and ORF predictions;521
11.2.4.3;4.3. Gene sets and orthogroup projections;521
11.2.4.4;4.4. Copy-number variation profiles;524
11.2.5;5. Analysis of Paralogous Genes;531
11.2.5.1;5.1. Estimating functional divergence between paralogous genes;531
11.2.5.2;5.2. Estimating divergence based on degree of conserved interactions;532
11.2.6;6. Discussion and General Applicability;533
11.2.7;References;535
11.3;Chapter 19: Experimental Evolution in Yeast: A Practical Guide;540
11.3.1;1. Introduction;541
11.3.2;2. Experiment Rationale;541
11.3.3;3. Experimental Evolution Approaches;543
11.3.3.1;3.1. Serial dilution;543
11.3.3.2;3.2. Chemostats;544
11.3.3.3;3.3. Turbidostats;545
11.3.3.4;3.4. More specialized systems;546
11.3.3.5;3.5. Miniaturization;546
11.3.4;4. Experimental Design;546
11.3.4.1;4.1. Growth conditions;546
11.3.4.2;4.2. Population size;547
11.3.4.3;4.3. Experiment duration;548
11.3.5;5. Practical Considerations;549
11.3.5.1;5.1. Strains and markers;549
11.3.5.2;5.2. Media;550
11.3.5.3;5.3. Growth rate;551
11.3.5.4;5.4. Good sterile practices;552
11.3.5.5;5.5. Good strain hygiene;552
11.3.5.6;5.6. Record-keeping;553
11.3.6;6. Analysis Techniques;553
11.3.6.1;6.1. Sampling regimen;553
11.3.6.2;6.2. Population genetics;554
11.3.6.3;6.3. Fitness;554
11.3.7;7. Example Protocol;555
11.3.7.1;7.1. Medium formulation;555
11.3.7.2;7.2. Chemostat preparation;555
11.3.7.3;7.3. Chemostat assembly;556
11.3.7.4;7.4. Inoculation;556
11.3.7.5;7.5. Daily sampling;556
11.3.7.6;7.6. Weekly sampling;556
11.3.7.7;7.7. Analysis;557
11.3.8;8. Conclusions;557
11.3.9;Acknowledgments;558
11.3.10;References;558
11.4;Chapter 20: Enhancing Stress Resistance and Production Phenotypes Through Transcriptome Engineering;562
11.4.1;1. Introduction;563
11.4.2;2. Transcription Factor Selection;564
11.4.3;3. Plasmid Library Construction;565
11.4.3.1;3.1. Promoter selection;565
11.4.3.2;3.2. Random mutagenesis by PCR;568
11.4.3.3;3.3. Quantification of total sequence diversity and library maintenance;571
11.4.4;4. Assessment of Phenotypic Diversity;572
11.4.4.1;4.1. Determination of yeast transformation efficiency;573
11.4.4.2;4.2. Evaluation of mutant libraries;574
11.4.4.3;4.3. Calculation of phenotypic diversity;575
11.4.5;5. Selecting for Phenotypes of Interest;577
11.4.5.1;5.1. Creation and maintenance of yeast library;578
11.4.5.2;5.2. Basic selection on liquid versus solid media;578
11.4.5.3;5.3. Alternative selection strategies and postselection screening;579
11.4.6;6. Validation;581
11.4.7;7. Concluding Remarks;582
11.4.8;References;583
12;Section 5: Advances in Cytology/Biochemistry;586
12.1;Chapter 21: Visualizing Yeast Chromosomes and Nuclear Architecture;588
12.1.1;1. Introduction;589
12.1.2;2. Strain Constructions and Image Acquisition for Nuclear Architecture Analysis in Living Cells;590
12.1.2.1;2.1. Tagging chromatin in vivo with lac and tet operator arrays;590
12.1.2.2;2.2. Determining the position of the nucleus;593
12.1.2.3;2.3. Immobilizing cells for microscopy;593
12.1.2.4;2.4. Controlling temperature;595
12.1.2.5;2.5. Image acquisition set-ups;595
12.1.3;3. Data Analysis and Quantitative Measurements;600
12.1.3.1;3.1. Accurate determination of the 3D position of a tagged locus;600
12.1.3.2;3.2. Colocalization of a DNA locus with a subnuclear structure;604
12.1.3.3;3.3. Quantification of locus mobility;605
12.1.4;4. IF and FISH on Fixed Samples;610
12.1.4.1;4.1. Yeast strains and media;611
12.1.4.2;4.2. Antibody purification and specificity;611
12.1.4.3;4.3. Choice of fluorop;612
12.1.4.4;4.4. Protocol;613
12.1.4.5;4.5. Special notes;618
12.1.5;References;619
12.2;Chapter 22: Quantitative Localization of Chromosomal Loci by Immunofluorescence;622
12.2.1;1. Yeast Strain Construction;623
12.2.2;2. Immunofluorescence;625
12.2.3;3. Fixing Cells;626
12.2.4;4. Spheroplasting;627
12.2.5;5. Preparing Slides;627
12.2.6;6. Antibody Incubations;628
12.2.7;7. Mounting and Storage of Slides;628
12.2.8;8. Microscopy and Analysis;629
12.2.9;Acknowledgments;631
12.2.10;References;632
12.3;Chapter 23: Spinning-Disk Confocal Microscopy of Yeast;634
12.3.1;1. Introduction;635
12.3.2;2. Building a Spinning-Disk Confocal Microscope;636
12.3.2.1;2.1. Microscope base;638
12.3.2.2;2.2. Scanhead;638
12.3.2.3;2.3. Lasers and filters;638
12.3.2.4;2.4. Choice of objective;639
12.3.2.5;2.5. Cameras;643
12.3.2.6;2.6. Other hardware considerations;646
12.3.2.7;2.7. Software;647
12.3.2.8;2.8. System integration;647
12.3.3;3. Sample Preparation;647
12.3.3.1;3.1. Fluorescent tagging and choice of fluorescent protein;647
12.3.3.2;3.2. Minimizing autofluores;650
12.3.3.3;3.3. Mounting;651
12.3.4;Acknowledgments;653
12.3.5;References;653
12.4;Chapter 24: Correlative GFP-Immunoelectron Microscopy in Yeast;656
12.4.1;1. Introduction;657
12.4.2;2. Recent Advances in High-Pressure Freezing and Freeze-Substitution;658
12.4.2.1;2.1. High-pressure freezing;658
12.4.2.2;2.2. Freeze-substitution;658
12.4.3;3. How to Prepare Yeast by HPF/FS;659
12.4.3.1;3.1. Growing and concentrating yeast;660
12.4.3.2;3.2. High-pressure freezing;660
12.4.3.3;3.3. Freeze-substitution;661
12.4.3.4;3.4. Embedding;663
12.4.3.5;3.5. Sectioning;664
12.4.4;4. Immunolabeling;665
12.4.5;5. Conclusions;666
12.4.6;6. Protocols;667
12.4.6.1;6.1. Filtration and HPF;667
12.4.6.2;6.2. Freeze-substitution;668
12.4.6.3;6.3. Embedding;669
12.4.6.4;6.4. Anti-GFP immunolabeling;670
12.4.7;Acknowledgments;670
12.4.8;References;670
12.5;Chapter 25: Analyzing P-Bodies and Stress Granules in Saccharomyces cerevisiae;672
12.5.1;1. Introduction;673
12.5.2;2. Determining If a Specific Protein can Accumulate in P-Bodies or Stress Granules;675
12.5.2.1;2.1. Markers of P-bodies and stress granules;675
12.5.2.2;2.2. Preparation of samples;679
12.5.3;3. Monitoring Messenger RNA in P-Bodies;683
12.5.4;4. Determining If a Mutation/Perturbation Affects P-Body or Stress Granule Size and Number;684
12.5.4.1;4.1. Conditions to observe increases or decreases in P-bodies and stress granules;684
12.5.4.2;4.2. Interpreting alterations in P-body/stress granule size and number;686
12.5.5;5. Quantification of P-Body Size and Number;688
12.5.5.1;5.1. Semiautomated quantification of P-body size and number;689
12.5.5.2;5.2. Manual quantification of stress granule size and number;690
12.5.6;Acknowledgments;690
12.5.7;References;690
12.6;Chapter 26: Analyzing mRNA Expression Using Single mRNA Resolution Fluorescent In Situ Hybridization;694
12.6.1;1. Introduction;695
12.6.2;2. Probe Design;696
12.6.3;3. Probe Labeling;698
12.6.3.1;3.1. Materials;698
12.6.3.2;3.2. Protocol;699
12.6.3.3;3.3. Measuring labeling efficiency;699
12.6.4;4. Cell Fixation, Preparation, and Storage;700
12.6.4.1;4.1. Materials;701
12.6.4.2;4.2. Protocol;701
12.6.5;5. Hybridization;703
12.6.5.1;5.1. Materials;703
12.6.5.2;5.2. Probes used for the hybridization shown in Figs. 26.1 and 26.3;704
12.6.5.3;5.3. Protocol;705
12.6.6;6. Image Acquisition;706
12.6.6.1;6.1. Microscope (example);707
12.6.7;7. Image Analysis;707
12.6.8;8. Summary and Perspectives;710
12.6.9;Acknowledgments;711
12.6.10;References;711
12.7;Chapter 27: The Use of In Vitro Assays to Measure Endoplasmic Reticulum-Associated Degradation;714
12.7.1;1. Introduction;715
12.7.2;2. In Vitro ERAD Assays Using a Soluble Substrate, p?F;718
12.7.2.1;2.1. Materials;719
12.7.2.2;2.2. The in vitro degradation assay for p?F;722
12.7.2.3;2.3. The p?F retrotranslocation assay;724
12.7.3;3. In Vitro Assays for Integral Membrane Proteins that are ERAD Substrates;726
12.7.3.1;3.1. In vitro ubiquitination assay;726
12.7.3.2;3.2. Analysis of integral membrane protein retrotranslocation;729
12.7.4;References;730
12.8;Chapter 28: A Protein Transformation Protocol for Introducing Yeast Prion Particles into Yeast;734
12.8.1;1. Introduction;735
12.8.2;2. Purification of Bacterially Expressed Sup-NM;736
12.8.2.1;2.1. Purification of Sup-NM;736
12.8.3;3. Preparation of Different Conformations of In Vitro Sup-NM Amyloid;737
12.8.3.1;3.1. Melting temperature analysis of Sup-NM amyloid;738
12.8.4;4. Preparation of In Vivo Prions from Yeast;738
12.8.5;5. Preparation of Lyticase;739
12.8.6;6. Protein Transformation;739
12.8.7;7. Determination of Prion Conversion Efficiency and Prion Strain Phenotypes;741
12.8.8;Acknowledgments;745
12.8.9;References;745
12.9;Chapter 29: Overexpression and Purification of Integral Membrane Proteins in Yeast;748
12.9.1;1. Introduction;749
12.9.2;2. General Considerations;749
12.9.3;3. Protocol-Molecular Biology;750
12.9.4;4. Protocol-Cell Growth;752
12.9.5;5. Protocol-Membrane Preparation and Solubilization;752
12.9.6;6. Protocol-Protein Purification;754
12.9.7;7. Protocol-Protein Characterization;758
12.9.8;8. Conclusion;759
12.9.9;Acknowledgments;759
12.9.10;References;759
12.10;Chapter 30: Biochemical, Cell Biological, and Genetic Assays to Analyze Amyloid and Prion Aggregation in Yeast;762
12.10.1;1. Introduction;763
12.10.2;2. Methods;765
12.10.2.1;2.1. Detecting protein aggregation in yeast cells;765
12.10.2.2;2.2. Assays for prion behavior;772
12.10.3;3. Concluding Remarks;784
12.10.4;References;784
13;Section 6: Other Fungi;788
13.1;Chapter 31: Genetics and Molecular Biology in Candida albicans;790
13.1.1;1. Homozygous Gene Disruption in C. albicans;791
13.1.1.1;1.1. Homozygous gene disruption by fusion PCR;793
13.1.2;2. C. albicans DNA Transformation;796
13.1.2.1;2.1. Transformation buffers;797
13.1.3;3. C. albicans Total RNA Purification;798
13.1.4;4. C-Terminal Epitope Tagging in C. albicans;799
13.1.4.1;4.1. Primer design;800
13.1.4.2;4.2. PCR conditions;800
13.1.4.3;4.3. Transformation;800
13.1.4.4;4.4. Integration confirmation;801
13.1.4.5;4.5. SAT1 marker excision;801
13.1.4.6;4.6. Tag sequence confirmation;801
13.1.4.7;4.7. Schematic of the 13×myc tagging procedure;802
13.1.5;5. C. albicans Chromatin Immunoprecipitation;803
13.1.5.1;5.1. Chromatin immunoprecipitation protocol;803
13.1.5.2;5.2. Chromatin immunoprecipitation buffers;806
13.1.5.3;5.3. Strand displacement amplification of ChIP samples;807
13.1.5.4;5.4. Strand displacement amplification solutions;809
13.1.5.5;5.5. Dye coupling;809
13.1.5.6;5.6. ChIP-chip hybridization protocol (adapted from the Agilent oligo aCGH/chip-on-chip hybridization kit);810
13.1.6;References;811
13.2;Chapter 32: Molecular Genetics of Schizosaccharomyces pombe;812
13.2.1;1. Introduction;813
13.2.2;2. Biology, Growth, and Maintenance of Fission Yeast;814
13.2.2.1;2.1. Other media supplements—Phloxin B;814
13.2.2.2;2.2. Other media supplements—Adenine and low-Ade media;819
13.2.2.3;2.3. Storage of fission yeast;819
13.2.2.4;2.4. Growth in liquid media;819
13.2.3;3. Genetics and Physiology;820
13.2.3.1;3.1. Performing genetic crosses;821
13.2.3.2;3.2. Testing mating type and sporulation by iodine staining;822
13.2.3.3;3.3. Random spore analysis;823
13.2.3.4;3.4. Tetrad dissection;823
13.2.3.5;3.5. Bulk spore germination;824
13.2.3.6;3.6. Isolating diploids;825
13.2.3.7;3.7. Cell synchrony in fission yeast cultures;826
13.2.4;4. Molecular Analysis;829
13.2.4.1;4.1. Fission yeast plasmids;829
13.2.4.2;4.2. Integrations in fission yeast;831
13.2.4.3;4.3. Isolation and analysis of novel mutations;832
13.2.4.4;4.4. Transformation of DNA into fission yeast;832
13.2.4.5;4.5. Harvesting DNA, RNA, and protein from fission yeast;834
13.2.5;5. Cell Biology;838
13.2.5.1;5.1. Preparation and analysis of cell populations;838
13.2.5.2;5.2. Flow cytometry (whole cells);838
13.2.5.3;5.3. Flow cytometry (nuclear ‘‘ghosts’’);839
13.2.5.4;5.4. DNA and septum staining in fixed cells;840
13.2.5.5;5.5. Fission yeast whole-cell immunofluorescence;841
13.2.6;6. Conclusion;844
13.2.7;Appendix: Schizosaccharomyces pombe Online Resources;844
13.2.8;References;846
13.3;Chapter 33: Applying Genetics and Molecular Biology to the Study of the Human Pathogen Cryptococcus neoformans;850
13.3.1;1. Introduction;851
13.3.2;2. Serotypes, Strains, and Sequences;852
13.3.3;3. Life Cycle;853
13.3.4;4. Techniques for Basic Culture;855
13.3.4.1;4.1. Dominant drug selection markers;856
13.3.5;5. Basic Molecular Biology Techniques;857
13.3.5.1;5.1. Fusion polymerase chain reaction;857
13.3.5.2;5.2. Transformation;861
13.3.5.3;5.3. Colony PCR;865
13.3.5.4;5.4. Genomic DNA extraction;867
13.3.5.5;5.5. RNA extraction;868
13.3.5.6;5.6. Protein extraction for SDS–PAGE;870
13.3.6;6. Methods for Assaying Pathogenesis;870
13.3.6.1;6.1. Murine model of infection;870
13.3.6.2;6.2. Tissue culture;878
13.3.6.3;6.3. Assays for characterized virulence factors;879
13.3.7;7. Concluding Remarks;881
13.3.8;Acknowledgments;882
13.3.9;References;882
13.4;Chapter 34: The Fungal Genome Initiative and Lessons Learned from Genome Sequencing;886
13.4.1;1. Introduction: Yeast Genomes and Beyond;887
13.4.2;2. Computational Prediction of Genes and Noncoding Elements;894
13.4.3;3. Mechanisms of Genome Evolution;896
13.4.4;4. Genomic Potential for Sex;900
13.4.5;5. Gene Family Conservation and Evolution;901
13.4.6;6. Impact of Next-Generation Sequencing;902
13.4.7;7. Future Directions;904
13.4.8;Acknowledgments;905
13.4.9;References;906
13.5;Chapter 35: Ultradian Metabolic Cycles in Yeast;910
13.5.1;1. Introduction;910
13.5.2;2. Induction of Ultradian Cycles of Oxygen Consumption Using a Chemostat;911
13.5.2.1;2.1. Chemostat setup;912
13.5.2.2;2.2. Comments;913
13.5.3;3. Long-Period Cycles;914
13.5.4;4. Short-Period Cycles;915
13.5.5;5. Significance of Ultradian Cycles;916
13.5.6;Acknowledgments;918
13.5.7;References;918
14;Author Index;920
15;Subject Index;932
16;Color Plates;946